Received 2000-04-25 Accepted 2000-06-26

Corresponding author. Tel: 86-311-606-2490; Fax: 86-311-606-2490; E-mail: syho@263.net

Acta Physiologica Sinica

Oct. 2000, 52 (5), 435¡«439

Changes in heart rate, blood pressure and renal sympathetic nerve activity

induced by microinjection of capsaicin into area postrema in rats

XUE Bao-Jian, HE Rui-Rongª³

(Department of Physiology, Hebei Medical University, Shijiazhuang 050017)

Abstract: The effects of capsaicin microinjection into area postrema (AP) on mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were investigated in 36 anesthetized Sprague-Dawley rats. The results obtained are as follows. (1) Following microinjection of capsaicin (10 ¦Ìmol/L, 50 nl) into the AP, MAP, HR and RSNA were significantly increased from 12.34¡À0.53 kPa, 328.52¡À7.54 bpm and 100¡À0% to 15.17¡À0.25 kPa (P<0.001), 354.81¡À8.54 bpm (P<0.001) and 156.95¡À7.57% (P<0.001), respectively. (2) Ruthenium red (RR, 100 mmol/L, 0.2 ml, iv), a capsaicin receptor antagonist, significantly inhibited these effects of capsaicin. (3) Pretreatment with a NMDA receptor antagonist MK-801 (500 ¦Ìg/kg, 0.2¡¡ml, iv) also reduced these effects of capsaicin. The above results indicate that microinjection of capsaicin into AP induces excitatory effects on MAP, HR and RSNA, which are mediated by capsaicin receptors with glutamate involvement.

Key words: capsaicin; area postrema; mean arterial pressure; heart rate; renal sympathetic nerve activity; ruthenium red; MK-801

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ѧ¿Æ·ÖÀàºÅ: Q463; R331.3

Capsaicin, a pungent ingredient of hot pepper, is a specific activator of nociceptive sensory neurons with C and A¦Ä fibers[1]. Its cardiovascular effects have been studied extensively[2,3]. Our previous study showed that intracarotid injection of capsaicin elicited increases in BP, HR and spontaneous discharge rate of nucleus paragigantocellularis lateralis (PGL) neurons[4]. The results suggested that capsaicin might exert an action on the brainstem nuclei involved in cardiovascular regulation.

Area postrema (AP) is a circumventricular organ located on the dorsal surface of medulla over the fourth ventricle and lacks a complete blood-brain barrier. AP neurons are susceptible to influences from circulating active agents as well as by afferent input from other brain regions. A large body of evidence implicats that AP plays an important role in the control of cardiovascular function[5,6]. Our previous study demonstrated that intracarotid injection of capsaicin resulted in an enhanced neuronal expression of Fos (a marker of neuronal activation) in brainstem nuclei involved in cardiovascular regulation including NTS, AP and PGL, specifically AP[7]. The possibility that central action of capsaicin is partly mediated through AP may be highly suggested. However, the direct effect of capsaicin on AP neurons has not yet been reported. The present study aimed to investigate the effects of microinjection of capsaicin into AP on BP, HR and renal sympathetic nerve activity (RSNA), and the possible underlying mechanism.

Sprague-Dawley rats weighing 300¡«350 g were anesthetized with urethane (1.0 g/kg, ip). The animals breathed spontaneously through a intratracheal tube. The right femoral artery and vein were cannulated for measurement of BP by a pressure transducer (MPU-0.5) and for drugs infusion, respectively. HR was monitored by a heart rate counter (AT-601G) triggered by differential signals of arterial pressure pulse. Method of bilateral buffer nerve section has been detailed in our previous study[6]. The left renal artery and vein were exposed via a retroperitoneal approach, and a branch of the renal nerve was isolated and clamped distally to eliminate the afferent activity. The nerve was placed on a bipolar platinum electrode for action potential recording, and immersed in liquid paraffin. The nerve activities were amplified by a biophysical amplifier (AB-621G) and then fed to an integrator (EI-601G), with an integrated time of 1 s. At the end of the experiments, the proximal end of the nerve was clamped to get the noise level of RSNA. The raw neurogram and the integrated RSNA as well as the BP and HR were recorded on a polygraph system (RM-6000, Nihon Kohden) with a thermal-array recorder (WS-682G, Nihon Kohden; band-pass width: 0¡«2.8 kHz). After midline incisions through the skin and epaxial musculature, the atlantooccipital membrane was punctured and a portion of the base of the skull removed with rongeurs. AP was clearly visible on the dorsal surface of the medulla. The animal was then placed in a stereotaxic frame (Model 1C, Jiang Wan) with its head in a nose-down position so that a glass micropipette (tip diameter 10¡«30 ¦Ìm) could be stereotaxically positioned on the dorsal midline surface of the AP and lowered ¡«200 ¦Ìm to AP injection sites. The injectio filled in the micropipette was delivered into the AP by a nanoliter injector (A203XVZ, World Precision, USA). The experimental animals were divided into the following groups: (1) A volume of 50 nl of a vehicle was injected into AP as the control for volume. After 30 min, capsaicin (10 ¦Ìmol/L, 50 nl) was microinjected into AP and the changes in BP, HR and RSNA were examined. (2) After iv injection of 0.2 ml saline was performed as vehicle control, a capsaicin receptor antagonist ruthenium red (RR, 100 mmol/L, 0.2 ml) was administered via venous route. After 5 min, microinjection of capsaicin into AP was carried out, and BP, HR and RSNA were examined. (3) BP, HR and RSNA were recorded following microinjection of capsaicin into AP before and after iv administration of a NMDA receptor antagonist MK-801 (500 ¦Ìg/kg, 0.2 ml). To identify the site of microinjection at postmortem, 2% pontamine sky blue was added to the injectio. At the end of the experiments, the rats were killed by decapitation, the brainstem was removed from skull and fixed in 10% formalin solution. After 7¡«10 d, frozen brain tissue was sectioned in the coronal plane (40 ¦Ìm). Histological verification was carried out with reference to Paxinos and Waton¡äs coordinates[8]. The stained area or the depth of the injecting track in AP was examined under the microscope. Data from those electrode tips not in the desired area were excluded.

Capsaicin, RR and MK-801 were purchased from Sigma. Capsaicin was dissolved in normal saline containing 10% ethanol and 1% Tween-80 and then diluted to final concentration with saline. The capsaicin receptor antagonist RR and NMDA receptor antagonist MK-801 were dissolved in normal saline.

All data were expressed as means¡ÀSE. The differences in the parameters between the vehicle and capsaicin were compared by paired Student¡äs t test. Differences between groups were assessed using unpaired t test. Statistical significance was accepted when P<0.05.

The results are as follows.

A total of 50 rats were used in the present study, of which 36 had histologically confirmed injection sites in AP. Figure 1 showed the medullary coronal section summarizing the locations of AP sites for capsaicin microinjection.

1.Effects of microinjection of capsaicin into AP on BP, HR and RSNA (n=20)

Microinjection of vehicle into AP caused insignificant changes in BP, HR and RSNA. Capsaicin (10 ¦Ìmol/L,

Fig.1.Coronal section of the dorsal medulla, illustrating histologically verified sites of capsaicin microinjection. AP, area postrema; NTS, nucleus tractus solitarius; CC, central canal£®50 nl) microinjected into AP evoked increases in BP, HR and RSNA, and the effects occurred within 80 s and lasted for 10 min (Fig.2 and Table 1).

2.Effects of RR on the actions of capsaicin (n=8)

Pretreatment with iv injection of RR significantly attenuated the effects of capsaicin on BP, HR and RSNA (Fig.2 and Table 1), whereas RR per se did not exert any actions on BP, HR and RSNA.

Fig.2.Effects of microinjection of capsaicin into AP on BP, HR and RSNA before and after iv administration of RR.

A. ¡ý, capsaicin. B. -¡¼¡½¡ýRR£«¡ý, capsaicin.

3.Effect of MK-801 on the actions of capsaicin (n=8)

Following iv injection of MK-801 (500 ¦Ìg/kg), the excitatory actions of capsaicin on BP, HR and RSNA were significantly inhibited (Fig.3 and Table 1), although MK-801 per se did not exert any action on BP, HR and RSNA.

The present study demonstrated that microinjection of capsaicin into AP induced increases in BP, HR and RSNA. Pretreatment with RR or MK-801 significantly suppressed the effects of capsaicin. The results suggested that capsaicin might activate the neurons of AP through vanilloid receptor (capsaicin receptor), and that glutamate might mediate the excitatory effects of capsaicin.

Table¡¡£±.Inhibitory effects of ruthenium red (RR, 100 mmol/L, 0.2 ml) or MK-801 (500 ¦Ìg/kg, 0.2 ml) on the responses of MAP, HR and RSNA to microinjection of capsaicin (Cap, 10 ¦Ìmol/L, 50 nl) into AP

[]n[]MAP (kPa)[]HR (bpm)[]RSNA (%)Control[]20[]12.34¡À0.53[]328.52¡À7.54[]100¡À0Cap[]20[]15.17¡À0.25***[]354.81¡À8.54***[]156.95¡À7.57***Control[]8[]12.55¡À0.54[]326.88¡À8.68[]100¡À0Cap[]8[]15.43¡À0.57***[]353.75¡À7.13***[]158.88¡À7.25***RR+Cap[]8[]13.68¡À0.29*##[]339.37¡À8.03**##[]123.75¡À5.95**###Control[]8[]12.91¡À0.27[]331.43¡À8.03[]100¡À0Cap[]8[]15.31¡À0.56***[]350.00¡À7.94***[]160.63¡À8.64***MK-801+Cap[]8[]13.28¡À0.42*###[]338.58¡À8.21**###[]133.13¡À5.08**###**P<0.001, *P<0.01, P<0.05, compared with control groups. ¡¡###P<0.001, ##P<0.01, compared with capsaicin groups.

Fig.3.Effects of microinjection of capsaicin into AP on BP, HR and RSNA before and after iv administration of MK-801.

A. ¡ý, capsaicin. B. -¡¼¡½¡ý MK-801 +¡ý capsaicin.

Recent studies have demonstrated that the effects of capsaicin on sensory neurons are mediated by capsaicin receptor[9]. Two specific antagonist of capsaicin receptor, capsazepine and RR, have been recently developed. Capsaicin receptors were found in AP in several species by [3H]resiniferatoxin (RTX) autoradiography[10], while our result that pretreatment with RR significantly attenuated the effects of capsaicin electrophysiologically revealed the presence of capsaicin receptors on AP neurons. Furthermore, since RR does not interfere with the capsaicin-binding site of the receptor, but blocks the capsaicin-activated ion channels, it is likely that the mode of action of capsaicin on AP neuron is similar to that reported from capsaicin sensitive afferent nerves, i.e. a depolarization induced by capsaicin receptor mediated the opening of a non-selective cation channel[9].

Both in vivo and in vitro experiments have demonstrated that capsaicin evoked the release of glutamate from central terminals of afferent fibers[11,12]. Sensory fibers have also been shown to terminate within the AP. A more specific neurotransmitter role for glutamate in the AP-NTS pathway was suggested by its localization in AP nerve terminals[13] and by recent study documenting that microinjection of glutamate into the AP elicited pressor and tachycardiac responses in rats[14]. Furthermore, central sensitization resulting from capsaicin treatment is prominently mediated by glutamate and NMDA receptor activation[15,16]. The result of the present study was in agreement with these findings.

AP is known to project to different regions of the brain including NTS, dorsal motor nucleus of vagus, parabrachial nucleus and RVLM. All these structures have also been implicated in autonomic regulation. Evidence for the role of the AP in modulating sympathetic activity was demonstrated by the finding that electrical stimulation of AP caused changes in the activity of neurons in RVLM[17], a site believed to be an important target for AP modulation and responsible for the generation of sympathetic activity[18]. In the present study, increased BP, HR and RSNA were seen during microinjection of capsaicin into AP. On the basis of the aforementioned reports and our results, it is conceivable that AP influences the cardiovascular system through these anatomical connections.

In summary, microinjection of capsaicin into AP elicits the increases in BP, HR and RSNA, which is an effect mediated by capsaicin receptor with the involvement of glutamate.

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